Red blood cell (RBC) alloimmunization remains a major life-threatening complication of transfusion. However, even the most immunogenic non-ABO antigen, RhD, alloimmunization rates do not reach 100%. Several factors including the host s immune status have been shown to influence the recipient s ability to react to RBC alloantigens, but it is not clear whether there may be differences in the immunological profile of those who will or will not develop transfusion-associated RBC alloantibodies. Increasing evidence indicates that CD4+ regulatory T cells (Tregs) characterized by co- expression of CD25 and the forkhead/winged helix transcription factor(Foxp3) are key suppressors of immunopathological immune responses. We have recently found that manipulation of Treg numbers affects the rate and the frequency of RBC alloimmunization in mouse models. Moreover, our preliminary results indicate that Treg suppressor activity is reduced in alloimmunized, transfused mice (responders) compared to non-alloimmunized, transfused recipients (non-responders). Furthermore, we have found that the effector cells from responders are resistant to Treg suppression. We will test the hypothesis that naive T cells upon encounter with the red cell alloantigen will either convert to become inducible Tregs (in non-responders) or differentiate into pathogenic effector cells (in responders) resistant to suppression. Using a novel model of chronic transfusion protocol in which half of the recipient mice develop antibodies against the transfused allogeneic red cells (responders), we will phenotypically and functionally characterize in detail Tregs and effector T cells to determine the molecular correlates of Treg suppressive activity in transfusion tolerant (non-responders) and intolerant (responders) mice. In parallel, we will also perform adoptive transfer studies using cells from the GPF-Foxp3 mice and compare the induction of Treg and effector cells by a combination of flow cytometry, and assays for suppression. The effect of manipulating the inflammatory milieu on Treg induction will be assessed and the data will be correlated to alloimmunization frequency. Altogether, we believe that our studies will be important for future identification of molecular signatures of "responders" such that phenotype-matched units can be pre-selected for this patient group, thereby reducing alloimmunization-associated morbidity and mortality. PUBLIC HEALTH RELEVANCE: Patients who are given life-saving blood transfusions can sometimes reject the transfused blood, which can result in death. The data from our animal studies suggest that if immune cells are not properly functioning, they will not accept transfused blood. The goal of our studies is to be able to determine in advance which patients may reject the transfused blood. This determination will allow patients to receive better care and avoid life threatening complications. In our grant application, we will expand our animal studies to focus on the triggers that make immune cells accept or reject transfused blood.